Project Summary/Abstract Dysregulation in gene expression is involved in most human diseases including cancers, developmental defects, and viral infections. Many therapeutic tools target defects in gene expression. The PI will study a key step in gene regulation ? 'how nascent triphosphorylated RNAs (ppp-RNAs) are regulated?'. RNA interference (RNAi) plays important roles in regulating genes, including viral and 'harmful' genes in all domains of life. This project will systematically examine how a highly conserved RNA polyphosphatase, C. elegans PIR-1, regulates gene expression by modifying ppp-RNAs in RNAi and non-RNAi-mediated pathways. In textbooks, nascent RNAs are synthesized as ppp-RNAs, which, as gene expression intermediates, are immediately converted to capped RNAs for protein expression or other functions. This study will demonstrate how an RNA polyphosphatase can change the fate of nascent ppp-RNAs, thereby modifying the textbook model. This gene regulation mechanism will serve as a new tool to regulate genes in research and therapeutics. Previous studies have indicated that human PIR-1 (hPIR1), a novel RNA polyphosphatase, removes two phosphates from ppp-RNAs to generate p-RNAs in vitro, which are then degraded. This activity renders PIR-1 a candidate for regulating ppp-RNAs including nascent mRNAs and viral RNAs. Consistently, previous studies suggested hPIR1 may be involved in regulating specific genes critical for cell proliferation in normal and tumor cells; however, these studies have provided few insights into: 1) what are the targets genes of PIR-1; 2) how PIR-1 works in vivo. The PI has identified two types of RNA substrates for C. elegans PIR-1, viral RNAs and endogenous mRNAs, both of which are silenced by PIR-1-mediated RNAi pathways. These preliminary results are consistent with the previous observation that PIR-1 may interact with Dicer, a central component of RNAi. Although the mechanism of RNAi has been extensively examined, previous studies have provided few insights into whether the ppp group on viral and cellular double-stranded (ds)RNAs plays any role in RNAi. The PI proposes PIR-1 may modify ppp-dsRNAs to generate p-dsRNAs, promoting Dicer to efficiently cut dsRNAs in C. elegans. The PI will systematically dissect the molecular mechanism of how PIR-1 regulates genes in these RNAi processes. The PI will also examine how PIR-1 regulates nascent ppp-RNAs in non-RNAi processes which play essential roles in the fertility of C. elegans. C. elegans is a robust genetic system especially in RNAi fields, and studies using C. elegans have led to several breakthroughs including three Nobel prizes in the last decade. The proposed research is original and innovative since it examines an underexplored yet key gene regulation mechanism. This research may lead to the development of more effective tools for regulating viral and endogenous genes. This project will have a significant and long-lasting impact on broad scientific inquiries including RNA biology, virology, developmental biology, and cancer biology. This study will also provide a new gene regulation tool for anti-cancer and anti-viral therapies, thus exerting a significant impact on public health.